One of the earlier mandatory steps in the carcinogenesis pathway is the tumor promotion event which allows for the clonal expansion of neoplastic cells. The main focus of this project is to define the potential autocrine/paracrine mechanisms regulating the growth of human tumors and utilize these as rational targets for early detection and intervention of malignant disease. Recently, we have demonstrated that the newly identified hypotensive peptide, adrenomedullin (AM), plays an important role in the proliferative process associated with carcinogenesis, embryogenesis and wound repair. AM has been shown to function as a mitogen, apoptosis survival factor and an angeiogenic factor, all of these characteristics playing a critical part in the in vivo proliferative process. Our prior studies have shown that a variety of human cancer cell lines and tumors have the ability to expressing AM and the AM-receptor (AM-R) which implicates these peptide/receptor partners in a possible autocrine growth mechanism regulating human neoplasm proliferation. We have demonstrated that AM expression in these tumor cell lines is upregulated during oxygen deprevation, an hypoxic state which normally exists in solid human tumors. Our studies have revealed that Hypoxia Inducible Factor-1 (HIF-1) act as the transcription factor which modulates AM expression under reduced oxygen tension by binding to hypoxia respnse elements at the 5? region of the AM gene. This was confirmed using luciferase report constructs transfected into MCF-7 followed by exposer to hypoxic conditions. In addition, cell lines derived from HIF-1 knockout mice which have a greatly reduced or complete absence hypoxia induced AM response. In addition, we have shown that reduced oxygen increased the half-life of the AM message going from 1.6 hours during normoxia to 2.5 hours for hypoxic conditions. Previously, we demonstrated the ability of a neutralizing anti-AM monoclonal antibody (MoAbG6) to block tumor cell growth in vitro and in vivo. In an attempt to find naturally occuring regulators of AM function, we have identified a 120,000 MW serum binding protein in a variety of mammalian species which selectively couples to AM. This interaction was shown to be specific for the intact native molecule and not for fragmentary AM products (AM1-12, AM13-52, AM16-21, AM22-52, AM34-52) or structurally related peptides (amylin or CGRP). Using a labeled ligand western blot technique in combination with preparative HPLC we have purified this 120,000MW AM binding protein, desginated as AMBP-1, to homogeneity and identified it to be human complement factor-H (factor H) by total amino acid compositions analysis and N-terminal amino acid sequencing via Edman degradation. Factor H is a naturally occurring serum protein which, when complexed with factor I, degrades C3b and blocks the complement cascade pathway of cell lysis. We have shown that commercially available factor H can block AM mediated antimicrobial effects on bacterial pathogens, however, it enhanced AM induced cAMP production in rat and mouse fibroblast cells in a dose dependent manner. We have also demonstrated that AM can augment the proteolytic degradation of C3b generated by the factor H/factor I complex. Factor H expression was identified in several human tumor cell lines (including carcinomas of the lung, breast, colon, ovary, prostate and skin) by northern and western blot analysis. Immunohistochemical examination of pathological specimens of human tumors with commercially available polyclonal antibodies to factor H have demonstated intense staining of neoplastic tissue. Thus the AM/factor H interactive link may be a novel promotion mechanism of carcinogenesis whereby tumor cells can circumvent immune surveillance by suppressing complement mediated lysis. The ability of MoAb-G6 to bind AM and sterically interfere with ligand/receptor recognition was the experimental basis to use this interaction to identify potential small molecule antagonists. In collaboration with Edward Sausville's group from the Developmental Therapeutics Program at the NCI, we have screened over 2,000 small molecules for their ability to disrupt AM/MoAb-G6 interaction and further examined these candidates to inhibit AM mediated cAMP production in Rat-2 fibroblast cells. Using this initial screening strategy we have identified twenty potential small molecule antagonists that will be further examined for their anti-growth effects on tumor cell proliferation. On-going studies with Drs. Kleinman, Kaur and Stetler-Stevenson using rat/chick aortic ring assays and a nude mouse capillary implantation model have confirmed AM to be an angiogenic factor capable of driving neovascularization in vitro and in vivo. Finally, we have shown that over-expressing AM in the human breast cancer cell lines MCF-7 and T47D induces a dramatic increase in the tumorigenicity of these lines. When comparing stable transfectants of AM over-expressors to empty plasmid control cells, the enhanced AM producing clones were resistant to apoptosis mediated by serum-deprivation, caused aortic ring outcropping in 24 hours and formed tumors in nude mice while the parent (or empty plasmid) control were unable to perform similar tasks.